Optimized MOL‐PCR for Characterization of Microbial Pathogens

Véronique Wuyts1, Nancy H.C. Roosens1, Sophie Bertrand2, Kathleen Marchal3, Sigrid C.J. De Keersmaecker1

1 Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health (WIV‐ISP), Brussels, 2 National Reference Centre for Salmonella and Shigella, Bacterial Diseases Division, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV‐ISP), Brussels, 3 Department of Information Technology, Ghent University, IMinds, Ghent
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.15
DOI:  10.1002/0471142956.cy1315s75
Online Posting Date:  January, 2016
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Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation‐PCR (MOL‐PCR) optimized for characterization of microbial pathogens. With MOL‐PCR, different types of markers, like unique sequences, single‐nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex‐TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL‐PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL‐PCR assay for subtyping of Salmonella Typhimurium. © 2016 by John Wiley & Sons, Inc.

Keywords: MOL‐PCR; Luminex; xTAG technology; microsphere suspension array

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Table of Contents

  • Introduction
  • Basic Protocol 1: Multiplex Oligonucleotide Ligation‐PCR
  • Support Protocol 1: Probe Design
  • Support Protocol 2: DNA Isolation
  • Support Protocol 3: Data Interpretation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Multiplex Oligonucleotide Ligation‐PCR

  • Ice or frozen cooling block
  • DNA lysate of positive control microbial isolates with known MOL‐PCR profile (see protocol 3)
  • DNA lysate of microbial isolates to be characterized (see protocol 3)
  • Multiplex ligation mix (see recipe)
  • Nuclease‐free distilled water
  • PCR mix (see recipe)
  • Microsphere mix (see recipe)
  • Reporter mix (see recipe)
  • 96‐well PCR plate (or PCR tubes) with compatible sealing
  • Vortex mixer
  • Microseal “A” film (Bio‐Rad)
  • Mini‐centrifuge for 96‐well plates or PCR tubes
  • Mini‐centrifuge for 0.5 ml microcentrifuge tubes
  • Thermal cycler
  • Luminex platform (a MAGPIX platform was used by the authors)
  • Bath sonicator
  • 96‐well plate with v‐bottom, compatible with the used Luminex platform
  • DynaMag‐96 Side Magnet (Life Technologies)

Support Protocol 1: Probe Design

  • Sequences of relevant molecular markers can be downloaded from Genbank (http://www.ncbi.nlm.nih.gov/genbank) or other sources
  • Anti‐TAG sequences are provided in the MagPlex‐TAG microsphere product information sheet, which can be downloaded from http://www.luminexcorp.com
  • For the design of MOL‐PCR probes, the authors have used Visual OMP software (DNASoftware); however, other software can be applied: Examples of other software are AlleleID (PREMIER Biosoft) or MOLigoDesigner (Song et al., )

Support Protocol 2: DNA Isolation

  • Culture of microbial isolate
  • Luria Bertani agar plate, sterile
  • Deionized water, sterilized by autoclaving
  • 37°C incubator
  • 1.5‐ml microcentrifuge tubes, sterile and compatible with heating block
  • Sterile, disposable 1‐μl inoculation loops
  • Vortex mixer
  • Heating block, capable of operating at 100°C
  • Centrifuge with microcentrifuge tube‐adapted rotor
  • 0.5‐ml microcentrifuge tubes or 96‐well plate with sealing for storage of DNA lysate at −20°C

Support Protocol 3: Data Interpretation

  • R software (R Core Team, ) is recommended for all calculations, but also an Excel workbook can be applied; however, the numeric precision of an Excel workbook may be too limited for calculation of large GPPs
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Literature Cited

Literature Cited
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